The objective of this project is to determine the mechanisms by which hydrostatic pressure and strain affect chondrogenesis by bovine chondrocytes in vitro. The mechanisms by which biomechanical stimuli influence signal transduction pathways and biomechanical regulation of the structure/function of the cartilage matrix, and metabolic function are not fully understood. The hypothesis of this research program is that application of specific biomechanical stimuli such as continuous or intermittent hydrostatic pressure, and micro strain alters accumulation of extracellular matrix, cytosolic second messengers, ion flux, reactive oxygen synthesis, and the anabolic activity of chondrocytes. In order to test this hypothesis, a series of specific aims have been developed, utilizing a validated 3D sponge culture system and methods in cell biology, biochemistry, molecular biology, and biomechanics. In this project, I plan to use an in vitro 3D sponge device and a pressure/perfusion system as a direct method to determine the effects of biomechanical stimuli on chondrogenesis. This novel pressure/perfusion system allows evaluation of matrix production by chondrocytes in a 3D collagen sponges under hydrostatic pressure of approximating physiologic levels. In addition, combined stimuli of hydrostatic pressure and microstrain will be tested with our system. Successful achievement of these goals could lead the way to the molecular characterization of precise molecular mechanisms by which biomechanical stimuli affect the signal transduction pathway in different types of cartilage. Furthermore, the proposed research may provide a new theoretical framework for cell-based treatment of cartilage disorders.